Myxococcus xanthus is an excellent prokaryotic system for studying the regulation of development. M. xanthus show a developmental cycle which is similar to the eukaryotic slime molds such as Dictyostelium discoideum. However, there are several distinct advantages to studying development in M. xanthus, since it is a simple gram-negative bacterium. We propose to study morphogenesis and the molecular mechanisms of control of gene expression during development using M. xanthus as a model system. We have purified and crystallized a development-specific protein, protein S. On the basis ot its partial amino acid sequence, the gene for protein S has been cloned with use of mixed synthetic oligonucleotide probes. The DNA sequence of the gene will be determined to elucidate how the gene is organized and regulated. Expression of the cloned protein S gene will be attempted both in E. coli as well as M. xanthus systems. For this purpose, multipurpose expression cloning vehicles developed in this laboratory will be extensively used. Second, the mechanism of secretion and assembly of protein S will be investigated using the cloned gene for protein S in E. coli as well as in M. xanthus systems. Third, we will further improve the method of two-dimensional DNA electrophoresis developed in this laboratory to a general method so that this method can be applied to detect phase variation in bacteria. Then the method will be used to detect if there is any phase variation during differentiation and morphogenesis. Fourth, we will continue to work to identify and isolate the origin of DNA replication with various methods, including synchronizing the initiation of DNA replication, restriction mapping a region of 100 to 150 kb containing the origin of DNA replication, and visualizing under the electron microscope the restriction fragments containing the origin. Functional assay of the origin will be attempted by constructing a plasmed containing the M. xanthus origin and examining its ability to duplicate in M. xanthus. The plasmid which we so construct will be used as a cloning vector for studying the expression of M. xanthus genes in M. xanthus. For this purpose, a transformation system for M. xanthus will also be developed.